Canola (Brassica napus) is one of the most economically important oilseed crops in Canada. Fusarium seedling blight is a root disease with the potential to cause severe yield reductions in canola. Fusarium spp. are commonly isolated root pathogens from fields in Alberta. Fusarium infection can also cause root rot in adult plants. In this study, 128 isolates identified as Fusarium spp. were recovered from field soils in central Alberta and from the roots of diseased canola plants with typical Fusarium seedling blight symptoms. Six species of Fusarium were identified, with Fusarium acuminatum as the predominant species (57 of 128 isolates, 44.5%). Phylogenetic analyses based on the translation elongation factor 1-α and the internal transcribed spacer sequence data were used for evaluation of genetic variations, and also used for Fusarium spp. identification in combination with morphological characteristics and polymerase chain reaction-based analyses. Based on disease ratings in pathogenicity tests, six isolates of F. avenaceum showed high aggressiveness on canola. Also, the aggressiveness varied within all Fusarium spp. No correlation was observed between aggressiveness and the geographic origin of the isolates.